Natural Killer (NK) cells are a major lineage of human lymphocytes with vital functions in innate and adaptive immunity and adaptive immunity to hematologic cancers. NK cell functions are mediated by the diverse interactions of highly polymorphic HLA-A, -B, and -C molecules with equally polymorphic killer immunoglobulin- like receptors (KIR). They are complemented by the conserved interactions of HLA-E with CD94:NKG2 heterodimers. The genes encoding KIR receptors and HLA ligands segregate independently, thereby generating unique and diverse genotypes within families and populations. Phenotypically, this produces functionally distinct NK cell repertoires, a natural variation that has profound effects on the outcome of HCT. To date, there has never been an appropriate in vivo xenogeneic model for studying NK-cell development and education through HCT or for validating the adoptive transfer of human NK cells. Historically, there have been two major barriers to study human NK-cell engraftment and function: (1) human NK-cell reconstitution and survival is dependent upon common ?-chain cytokines IL-2, IL-7 and IL-15 that are not cross reactive between species; and (2) human NK-cell function is educated, regulated and maintained by inhibitory receptor engagement with MHC class I molecules that are also not cross-reactive between species. We have now overcome the cytokine and MHC class I barrier by utilizing a cutting-edge adeno-associated virus (AAV) vector-mediated gene delivery approach to transduce genes encoding HLA-A, -B, -C and ?E polypeptides, as well as certain human cytokines, to highly immunodeficient NSG mice that lack mouse-derived ?2- microglobulin, named NSG-B2M-/- mice. In Specific Aim (SA)1 we will establish human immune system (HIS) mice expressing select human cytokines and HLA-A*02 and/or HLA-E. The cytokines IL-2, IL-3, IL-6, IL-7, IL- 15 and GM-CSF will promote development of human NK cells along with human T cells, B cells, myeloid macrophages and DCs. We will apply a 42-plex mass cytometry antibody panel to closely map lymphocyte reconstitution following HCT at an unprecedented resolution. NK cell development, education and function will be studied longitudinally over a range of 20 weeks. In SA2 we will infect HIS mice with luciferase-labeled tumor target cells in order to evaluate the impact of NK cell education on anti-tumor activity and outcome of HCT. We will compare anti-tumor function of NK cells from HIS mice by mass cytometry as well as tumor burden by bioluminescence imaging. Our HIS mouse model will provide a definitive answer on whether HLA-E can directly educate CD94:NKG2A+ human NK cells and how this education impacts their capacity to respond to circulating tumors. In SA2, we will also measure the in vivo effects of CMV infection (and reactivation) on the education of adaptive NK cells and their enhanced anti-tumor response. With the ultimate goal of developing HIS mice expressing human cytokines along with complex HLA class I haplotypes, we will be able to effectively harness NK cell effector for treatment of hematologic cancers.